Average volume digital computer and digital volume totalizer for cells and particles

ABSTRACT

An apparatus for deriving average volume and total volume data from particles in a flow of liquid metered through an orifice includes a display of mean particle volume and total particle volume and count. The mean particle volume is the quotient of the totalized particle volume of a specific number of particles divided by that same number. The total particle volume and count provided by the same apparatus can be used to confirm the mean particle volume measurement.

United States Pate Berg [451 Oct. 17, 1972 [54] AVERAGE VOLUME DIGITAL 13,259,842 7/1966 Coulter et al ..235/92 PC COMPUTER AND DIGITAL VOLUME3,626,164 12/1971 Pontigny et al. ...324/71 CP X TOTALIZER FOR CELLS AND3,557,352 l/l97l Hogg et al. ..235/l5 [.3 PARTICLES PrimaryExaminer-Eugene G. Botz Inventor; Robert g, 196 Cllntofl AssistantExaminer-Edward J. Wise Avenue, Elmhurst, Ill. 60126 Attorney-Hill,Sherman, Meroni, Gross & Simpson 22 F d: h 1 7 lie 7 Marc 0 l9 1 S ACT[21] Appl' An apparatus for deriving average volume and total volumedata from particles in a flow of liquid metered [52] 0.8. CI. ..235/l51.34, 235/92, 324/71 hr gh an orifice includes a display of meanparticle [51] Int. Cl ..G06f 15/20 volume and total particle volume andscum- The [53] Fi ld f s h 235/151 34 92 324 7 R mean particle volume isthe quotient of the totalized 324/71 CP; 356/39, 40, 102; 340/347Particle volume of a specific number of particles div vided by that samenumber. The total particle volume [56] References Cited and countprovided by the same apparatus can be used to confirm the mean particlevolume measure- UNITED STATES PATENTS 3,502,973 3/1970 Coulter et al...235/92 PC 6 Claims, 5 Drawing Figures 70 VACUUM Z4 OK/F/CE Ct/RRESUPPLY z; I a l uA/EAR 59 l AMPLIFIER Pl/456 AMPuruaE wolf/26k (40c) 26J0 l 227/ Acct/M0247 6/ l7 (MU/(2f PULSf SWTJR J3 Z i/ )2 l 7 Z,

| I I 7am cam/r new m menu! mun-0am) a/sPmY a min/m7 less/am: 1 K66lsIER Reels/EA AVERAGE VOLUME DIGITAL COMPUTER AND DIGITAL VOLUMETOTALIZER FOR CELLS AND PARTICLES BACKGROUND OF THE INVENTION It haslong been the practice in clinical laboratories to determine thefraction of cell volume in whole blood by centrifuging blood in a thin,constant diameter tube, and then manually positioning the tube adjacentto suitable scale to read the hematocrit percentage at the interfacebetween the dark red packed cells and the comparatively clear plasma.This procedure is primarily manually performed and is, therefore, a timeconsuming operation.

Another method for determining the percent of cell volume involves theuse of an orifice tube and orifice current supply in combination withvacuum apparatus well known in the art to generate electrical pulseswhich are proportional to particle volume. The pulses are operated upon,for example, by shaping to provide an integrated charge on a capacitorwhich is utilized to operate a motor mechanism for a scale pointer, pen

recorder or the like. Such procedure, however, suffers from changes inambientconditions and analog component stability and requires apparatusfor shaping the receivedpulses.

SUMMARY OF THE INVENTION Another object of this invention is to providedirectly for the case of blood analysis an improved method for measuringnot only the percentage of cell volume in whole blood (I-ICT) but theaverage cell volume (MCV), which may also be obtained by dividing the hematocrit percentage (I-ICT) by the red blood cell (RBC) count in a unitvolume of blood and multiplying by a power of 10.

, Another object of this invention is to provide the precision andstability of digitally computed parameters in a manner providing doublechecking, for example, by confirming the MCV by dividing the HCT by theRBC count and multiplying by a power of 10, and accomplishing theforegoing without resort to a programmed digital computer with theattendant cost of complex, sophisticated circuitry and software.

A further object of this invention is to provide relatively simple,accurate instrumentation for widely used applications which require thistype of particle volume analysis for large numbers of samples, such asclinicallaboratories for red blood cells.

An instrument apparatus, for example, scans a flow of 2,5 60,000 cubicmicrons of whole blood in a diluted state to generate a pulse for eachcell traversing an orifice such that the pulse amplitude is directlyproportional to the volume of the cell. These pulses are suitablyfactored to provide the direct RBC count.

For determining the I-ICT value, these pulses are also presented to ananalog to digital converted (ADC) which provides a digital pulse trainoutput for each cell such that each pulse of said train represents twocubic microns of cell volume. This pulse train from the ADC is thendivided by 12,800 for direct display on a counter as the I-ICTpercentage when the RBC count is completed. The apparatus includes meansfor producing start and stop signals for these displays which correspondto the initiation and termination of the scanned flow through theorifice, in addition to means for producing the individual particlepulses, for converting the amplitudes of said pulses to a digital pulsetrain, and for properly dividing the pulse train.

The apparatus, according to the present invention, is also provided withindependent means for measuring and displaying the MCV directly in cubicmicrons, by which the digital pulse train output of the ADC is dividedby a suitable factor 2,048 being an exemplary illustration, and thisdivided output is counted until I Stopped by a signal provided at acount of twice that number of cells, i.e., 4,096 cells. Again, eachpulse of said train means two cubic microns of cell volume; the totalvolume of the 4,096 cells thus being divided by 4,096 in an on-linemanner.

BRIEF DESCRIPTION OF THE DRAWINGS Other objects, features and advantagesof the invention, its organization, construction and operation will bebest understood from the following detailed description of an exemplaryembodiment thereof, taken in conjunction with the accompanying drawings,in

which: I I

- FIG. 1 is a circuit block diagramof particle volume analyzingapparatus according to the invention;

FIG. 2 is a circuit diagram of a pulse analog to digital converter whichmay be employed in the circuit illustrated in FIG. 1;

FIG. 3 is a graphical illustration of wave forms and event sequences atselected points in the circuit illustrated in FIG. 2;

FIG. 4 is a circuit representation of an accumulator which may beemployed in the circuit of FIG. 1; and

FIG. 5 is a graphical illustration of wave forms and event sequences atselected points in the circuit illustrated in FIG. 4.

DESCRIPTION OF THE PREFERRED EMBODIMENT An electric sensing zone 10comprises a container 11 holding therein conductive liquid.l2 containingparticles to be analyzed and an agitator l5 operable to maintain theparticles continuously suspended in the liquid 12. An orifice tube 13which is also filled with the liquid 12 is disposed in the container 11and. includes an orifice 14 to pass the liquid into the tube I3 in orderto establish an electrical circuit between an electrode 22 within thetube 13 and an electrode 23 disposed below the surface of the liquid 12in the container 11. The tube 13 includes an end for connection to asource of negative pressure, a vaccuurn supply, the application of suchnegative pressure being controlled by an interposed valve 19.

Also connectable to the source of vacuum by way of the valve 19 is amercury siphon tube 16 having an open end 18 and containing a supply ofmercury. Located within said siphon tube 16 is a pair of electrodes 20,21 for controlling the start'and stop operations of particle and totalvolume count, as will be explained in greater detail below.

The electrodes 22, 23 are connected by way of a pair of conductors to acurrent supply 24 to establish acurrent flow in the circuit includingthe electrodes 22, 23

as is well known in the art. Further, and as also known in the priorart, the passage of particles through the orifice modulates the currentflow during their passage to produce a sequence of voltage pulses. Thesepulses and. their amplitudes represent the particle population and sizeof particles inthe liquid 12.

The particle pulses are fed to a linear pulse amplifier 25 which has anoutput connected to an adjustable trigger. circuit 26 for triggering acount register 27 during the interval of engagement of the mercury 17with the start andv stop electrodes 20, 21, respectively. Reference maybe had to U.S. Pat. No. 3,345,502 for a more detailed discussion ofthistype of apparatus and its operation which will not be treated infurther detail herein. It suffices here to state only that the amplifier25 is provided with'particle pulses whose amplitude is directlyproportionalto particle volume and that there isan interval ofpulsecount corresponding to the contacting of the mercury with theelectrodes 20, 21 after the valve 19 to the regulated vacuum has beenclosed.

' The linear particle pulse amplifier 25 provides the particle pulses toa pulse amplitude digitizer 29 (ADC) which convertsthe amplitude of theparticlepulses into digital information signals. This digitalinformation is made available to an accumulator which operates as aparticle pulse summator in an overflow mode.

The accumulator 30 has a first output tapped off at an intermediatecount which represents in digital form the total volume of the particleswhich have traversed the orifice 14. This information is provided to atotal count volume register 31 which includes apparatus for displayingthe total volume. A second output of the-accumulator is connected to amean volume register 32 which is effective to register the mean particlevolume by summing overflows of the accumulator. As can be seen from thedrawing, the total-count volume register is operable. for a period oftime governed by the start and stop electrodes 20, 21; whereas the .meanvolume register has its start'control governedby the start electrode butits stopicontrol provided by way of a predetermined countstop apparatus33 which isdriven by the trigger circuit 26. The predetermined countapparatus 33 is provided as a factor arrangement whereby thepredetermined count may be evenly related to the volume of particles ona simple decimal multiple basis of the average particle volume.

In a particular system constructed which is capable of running a typicalRBC operation, the predetermined count from the apparatus 30 wasselected to be 4,096 and the overflow count was selected to occur at2,048 in that the apparatus. provides two counts per cubic micron. Theoutput to the total count volume register 31 was selected to occur ateach count of.128, this count being divided by 100 in the total countvolume register. Therefore, 128 pulses equals 256 cubic microns andtheir summation divided by 100 yields pulses meaning 25,600 microns.With blood diluted at 62,500 X 160 microliters of such a dilution equalsthe aforementioned 2,560,000 cubic microns as a sample of whole blood tobe scanned. Whilei reference is made hereinto conducting an RBCoperation, it is clearly evident that. the instant invention isapplicable for analyzing many other types of particle populations.

Referring .to FIGS. 2 and 3, the pulse amplitude digitizer 29 and itsoperation will be set forth. It should be noted that the lower casereference characters 0-): adjacent wave shapes in FIGS. 2 and 4correspond to the trace references in FIGS. 3 and 5 and arrows of thereference characters 0-! are influences or cause and effect references.The particle pulses a are received by the digitizer 29 on an inputconductor 34 and fed to an operational amplifier stage which isconstructed for unity gain. The operational amplifier stage includes anamplifier 35 having the conductor 34 connected toits positive input, adiode 36 serially connected to the output of the amplifier 35 and'afeed-back conductor 37 connecting the diode 36 to the negative input ofthe amplifier 35. A capacitor 38 is connected to the diodev 36 and iseffective to store energy in response toapar- 'ticle pulse, the voltageacross the capacitor representing the'amplitude of the particle pulse,and .iseffective to remember the pulse amplitude. The pulses received onthe conductor 44 are also connected to the positive input of anamplifier 40 which has its negative input connected to, a variableresistor 41 which is adjustable to set a threshold level or noisediscriminationrlever a so that the apparatus is rendered insensitive tolow level signals. The amplifier 40 operates to provide'an output pulsehaving a width determined by the crossings of the level a'by a particlepulse. This output signal c is connected to the set input of a flip-flop42 which is responsive to initiate an output pulse d for turning on aconstant current generator device 43 which includes. a switch 44 and aconstant current generator 45. The activation of the constant currentapparatus 43 is effec tive to discharge the capacitor 38 in accordancewith the expression T=C V, where I is the constant current, C is thecapacitance of the capacitor 38, V is the initial voltage across acapacitor "38 and T is the discharge time of the capacitor 38. With thecapacitance and the current being constant, the discharge time is shownto be linear. The resulting wave form b is applied to the positive inputof an amplifier 39 to terminate a pulse e which was initiated inresponse to the initial start of charging of the capacitor 38 as thevoltage thereacross went above zero.

The pulse e is fed to a differentiator circuit 46 which responds to thetrailing edge of the pulse 2 to provide a spike f for application to thereset input of the flip-flop 42 which causes the flip-flop to reset andtermination of the pulse d.

The output of the flip-flop 42 is also applied to one input of a ANDgate 47 which has another input connected to a clock (pulse generator)48. The pulse d is effective to gate the output of the clock through thegate 47 to a gate 49 which passes the digitized output signal h. Thepulse d itself is likewise-passed through a gate 53 as an availableinvented output signal d'.

In order to reject high amplitude pulses which are not within the rangeof sizes of particles being evaluated, for example a fiber in a bloodsample, an upper limit threshold level a is established by means of avariable resistance 51 connected to the negative input of an amplifier50. The positive input of the amplifier 50 is connected in common withthe positive input of the amplifier 40 to the input conductor 34. Theamplifier 50 has its output connected to a flip-flop 52. The amplifier50 produces a pulse j having a width determined by the crossing of theupper threshold a by a particle pulse. The pulse j is effective to setthe flip-flop 52 and initiate a pulse k' which is terminated upon resetof the flip-flop 52 by way of a pulse f which was initiated in responseto the discharge of the capacitor 38 as described above. The pulse k iseffective to block the gates 53 and 49 and prevent outputting of therespective signals d, h therefrom.

Referring now to FIGS. 4 and 5, the accumulator and its relation to thedigitizer 29 and to the volume display register 31 and 32 isillustrated. The output h is supplied by way of a conductor 54 to acounter 55 which divides the output h by a factor X, for example 2,048,and the output h/X is presented to a gate 56. The output d is appliedover aconductor 57 to a counter 58 where another factor Y, for example4,096, is applied at the resulting output m,(d'/ Y), is applied to thegate 56 to gate through the output I as a signal of h/X to the MCVdisplay register of 32 which continues to receive, register and displaythe signal 1 until receipt of a stop .signal from the predeterminedcount device 33, which occurs at, in this particular example, the countof 4096.

The counter 55 also applies a factor of Z to divide the signal h by, forexample, 128 to provide the signal h/128 as a pulse series n to thetotal count volume display register 31 which is operable to receive,register and display counts during the start-stop interval initiated atthe electrodes 20, 21 in FIG. 1. The counted signal h is thereforecounted for the entire aliquot of sample and yields the factored HCTreading.

Although i have described my invention by reference to a specificapplication of a particular embodiment thereof, many changes andmodifications of my invention may become apparent to those skilled inthe art without departing from the spirit and scope of the invention,and it is to be understood that I intend to include within the patentwarranted hereon all such changes and modifications as may reasonablyand properly be'included within the scope of my contribution to the art.

What I claim is:

1. ln apparatus of the type wherein a liquid suspension of particles iscaused to flow through a sensing zone, sensing means are provided togenerate electrical pulses in response to the individual particlestraversing the sensing zone for a predetermined interval, the particlepulses having amplitudes directly proportional to the volumes of theindividual particles, means are provided for counting the particlepulses over the predetermined interval, and a data converter is utilizedto register and display particle volume data in accordance with theamplitudes of the generated particle pulses, the improvement whereinsaid data converter comprises: 7 a pulse height analog to digitalconverter for converting the individual particle pulses into digitalsignals, means for summing said digital signals, means for summingsaiddigital signals, means operable over the entire predeterminedinterval for applying a first factor to the summation of said digitalsignals to obtain total relative 6 particle volume, and means operableover a predetermined number of particle pulses to apply a second factorto the summation of said digital signals to obtain mean particle volume.

2. The improvements set forth in claim 1, wherein said means for summingsaid digital signals includes an accumulator connected to said analog tod igital converter, and said means for applying a first actor and saidmeans for applying a second factor each include respective selectedoutput taps of said accumulator.

3. ln apparatus of the type wherein a liquid suspension of particles iscaused to flow through a sensing zone, sensing means are provided togenerate electrical pulses in response to the individual particlestraversing the sensing zone for a predetermined interval, the particlepulses having amplitudes directly proportional to the volumes of theindividual particles, means are provided for counting the particlepulses over the predetermined interval, and a data converter is utilizedto register and display particle volume data in accordance with theamplitudes of the generated particle pulses, the improvement whereinsaid data converter comprises: a pulse height analog to digitalconverter for converting the individual particle pulses into digitalsignals, means for summing said digital signals, and means operable overthe entire predetermined interval for applying a factor to the summationof said digital signals to obtain total relative particle volume.

4. The improvement set forth in claim 3, wherein said means for summingsaid digital signals includes an accumulator connected to said. analogto digital converter, and said means for applying a factor includes aselected output tap of said accumulator.

5. ln apparatus of the type wherein a liquid suspension of particles iscaused to flow through a sensing zone, sensing means are provided togenerate electrical pulses in response to the individual particlestraversing the sensing zone for a predetermined interval, the particlepulses having amplitudes directly proportional to the volumes of theindividual particles, means are provided for counting the particlepulses over the predetermined interval, and a data converter is utilizedto register and display particle volume data in accordance with theamplitudes of the generated particle pulses, the improvement whereinsaid data converter comprises: a pulse height analog to digitalconverter for converting the individual particle pulses into digitalsignals, means for summing said digital signals, and means operable overa predetermined number of particle pulses for applying a factor to thesummation of said digital signals to obtain mean particle volume. 1

6. The improvement set forth in claim 5, wherein said means for summingsaid digital signals includes an accumulator connected to said analog todigital converter, and said means for applying a factor includes aselected output tap of said accumulator.

mam-swa mmswm CETEFECATE or ConmtrieN i y Y Issue Patent No. 3, 699,319- Dated QCtObQI' 17 1972 Inventoflsj Robert Berg It is certified thaterror appears in the above-identified. patent and that said LettersPatent are hereby corrected as shown below:

IN THE SPECIFICATION:

Column 3, line 3 read "l5""as -14-3- Coiurnn 4, line 18 read "44" as-34-. v Column 4,. line 55, read "invented"as inverte d--.

IN THE CLAIMS:

Claim 1, lines 16 and 17 cancel "rneans for summing said digitalsignal-e," as being redundant with respect to the identical precedingwording. v V

Signed and sealed this 29th day of May 1973.

(SEAL) Attest:

EDWARD -M.'FLETCHER-,JR. ROBERT, GOTTSUCH'AL K Attestlng OfficerCommissioner of Patents FoRM P o-ioso (io-s's) I uscoMM-Dc wave-ps9 VRNMEHT PRINTING OFFICE I959 0-356-334

1. In apparatus of the type wherein a liquid suspension of particles iscaused to flow through a sensing zone, sensing means are provided togenerate electrical pulses in response to the individual particlestraversing the sensing zone for a predetermined interval, the particlepulses having amplitudes directly proportional to the volumes of theindividual particles, means are provided for counting the particlepulses over the predetermined interval, and a data converter is utilizedto register and display particle volume data in accordance with theamplitudes of the generated particle pulses, the improvement whereinsaid data converter comprises: a pulse height analog to digitalconverter for converting the individual particle pulses into digitalsignals, means for summing said digital signals, means operable over theentire predetermined interval for applying a first factor to thesummation of said digital signals to obtain total relative particlevolume, and means operable over a predetermined number of particlepulses to apply a second factor to the summation of said digital signalsto obtain mean particle volume.
 2. The improvements set forth in claim1, wherein said means for summing said digital signals includes anaccumulator connected to said analog to digital converter, and saidmeans for applying a first factor and said means for applying a secondfactor each include respective selected output taps of said accumulator.3. In apparatus of the type wherein a liquid suspension of particles iscaused to flow through a sensing zone, sensing means are provided togenerate electrical pulses in response to the individual particlestraversing the sensing zone for a predetermined interval, the particlepulses having amplitudes directly proportional to the volumes of theindividual particles, means are provided for counting the particlepulses over the predetermined interval, and a data converter is utilizedto register and display particle volume data in accordance with theamplitudes of the generated particle pulses, the improvement whereinsaid data converter comprises: a pulse height analog to digitalconverter for converting the individual particle pulses into digitalsignals, means for summing said digital signals, and means operable overthe entire predetermined interval for applying a factor to the summationof said digital signals to obtain total relative particle volume.
 4. Theimprovement set forth in claim 3, wherein said means for summing saiddigital signals includes an accumulator connected to said analog todigital converter, and said means for applying a factor includes aselected output tap of said accumulator.
 5. In apparatus of the typewherein a liquid suspension of particles is caused to flow through asensing zone, sensing means are provided to generate electrical pulsesin response to the individual particles traversing the sensing zone fora predetermined interval, the particle pulses having amplitudes directlyproportional to the volumes of the individual particles, means areprovided for counting the particle pulses over the predeterminedinterval, and a data converter is utilized to register and displayparticle volume data in accordance with the ampliTudes of the generatedparticle pulses, the improvement wherein said data converter comprises:a pulse height analog to digital converter for converting the individualparticle pulses into digital signals, means for summing said digitalsignals, and means operable over a predetermined number of particlepulses for applying a factor to the summation of said digital signals toobtain mean particle volume.
 6. The improvement set forth in claim 5,wherein said means for summing said digital signals includes anaccumulator connected to said analog to digital converter, and saidmeans for applying a factor includes a selected output tap of saidaccumulator.